Organic synthesis of chemical intermediates and raw materials important to the polymer and chemical industries can be a major portion of the cost of production of the finished product made from them. Often, starting materials are synthesized from petroleum products which are non-renewable, expensive, and may be hazardous. In addition, the cost of petroleum products is dependent on world markets and other factors which can not be controlled. Another factor which increases costs in the production of organic molecules by a chemical synthetic route is the resulting waste stream of unwanted byproducts and contaminated solvents, the disposal of which is expensive and hazardous. Yet, for most chemical starting materials and intermediates, there is no other practical alternative to chemical synthesis.
Production of starting materials by biofermentation is an alternative which uses renewable resources and creates much less hazardous waste. The fermentative production of a few chemicals has been reported (W. Crueger and A. Crueger, Biotechnology: A Textbook of Industrial Microbiology, Sinauer Associates, Sunderland, Mass., pp. 124-174 (1990); B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, second edition, Stockton Press, NY, pp. 243-364 (1991)) and commercialized. In a recent review on improving the production of aromatic compounds in Escherichia coli (E. coli) by metabolic engineering, an extensive array of compounds is detailed (A. Berry, Tibtech 14:250-256 (1996)).
Unfortunately, this is not a commercially feasible alternative for a great many desirable molecules because the molecule of interest is produced in such small amounts by the biocatalytic organisms. Even organisms genetically engineered to produce enhanced amounts of the desired molecule often do not produce concentrations of the product great enough to justify the investments necessary to develop a commercial biofermentation process. This can be particularly true when the product is toxic to the cells or is regulated by a negative feedback mechanism, thereby limiting the potential concentration of the product in the fermentation medium.
At the same time, one of the greatest challenges to the development of a biofermentative process is the separation, recovery, and purification of the product from the fermentation medium at relatively low concentrations and in the presence of other chemical species. Removal of the desired metabolites from fermentation mixtures is typically performed after the termination of the fermentation process. Generally, the process involves a first filtration step to separate the biomass and yield a clarified solution from which product is removed. Often, the process then includes an energy-intensive water-removal step to concentrate the desired product or products. The product of interest must then be separated from the other molecules in the fermentation medium, for example metabolic byproducts and culture medium constituents.
This separation and purification may be performed by any of a number of techniques known in the art of biochemistry, such as electrodialysis, electrophoresis, sedimentation, solvent extraction, precipitation, or distillation. For example, lactic acid may be purified from fermentation medium using electrodialysis (P. Vonktaveesuk et al., J. Ferment. Technol. 77:508-512 (1994); Y. Nomura et al., J. Ferment. Technol. 71:450-452 (1991) or extraction (V. Yabannavar and D. Wang, Biotechnol. Bioeng. 37:1095-1100 (1991)). Acetic acid has also been purified after biofermentation using electrodialysis (S. Shang and K. Toda, J. Ferment. Technol. 77:288-292 (1994)).
Standard techniques are not commercially viable unless the product is present in the fermentation broth in relatively high concentrations, therefore they are limited in usefulness to products which are produced in relatively high concentrations in the fermentation medium. Even when feasible to do in the laboratory, the product recovery steps add to manufacturing costs often preventing the commercialization of the fermentative production of a wide range of chemicals. For production of these compounds to be cost-effective, more selective and less expensive techniques must be developed to remove and purify low concentrations of product.
For example, 4-hydroxybenzoic acid (PHB), a key monomer in the synthesis of liquid crystalline polymers (LCPs) with additional utility as a chemical intermediate for the manufacture of paraben preservatives and other products, is currently produced by organic chemical synthesis and costs approximately $2.40/lb. The high cost of PHB contributes significantly to the high cost of LCPs and thus limits the applications to which LCPs can be put commercially. The biofermentative production of PHB from glucose can potentially reduce the manufacturing costs of LCPs now, and in the future by insulating PHB from potential increasing petrochemical costs as oil reserves are depleted. A key challenge in the fermentative production of this chemical, however, is the isolation of the desired product from the aqueous fermentation mixture at relatively low concentrations (&lt;10% w/v) in the presence of other chemical species (W. Crueger and A. Crueger, Biotechnology: A Textbook of Industrial Microbiology, Sinauer Associates, Sunderland, Mass., pp. 111-123 (1990); B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, second edition, Stockton Press, NY, pp. 905-1022 (1991)).
PHB can be made from glucose using E. coli, which produces the chemical as a minor metabolite and excretes the product into the medium at levels of less than 2 mg/L. The biosynthetic pathway in E. coli is shown in FIG. 1. A significant number of steps are required in the metabolic pathway from 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to chorismic acid, variously termed the chorismic acid, aromatic amino acid, or shikimic acid pathway.
Genetically engineered biocatalysts are known to produce chemicals like PHB. However, the amounts produced by these strains are not optimal for recovery by the prior art methods. There is a need, therefore, for a method to increase the recovery of PHB commercially produced using biofermentative methods.